Pneumocystis carinii is an opportunistic pathogen which causes pulmonary infections. In particular, P. carinii is a common causative agent of pneumonia in immunocompromised individuals, such as patients with AIDS, children with immunodeficiency disorders and those receiving immunosuppressive therapy for malignancy or transplantation. The rapid rise in the incidence of AIDS since the early 1980s has changed the status of P. carinii from a rare opportunistic pathogen to a common infection in immunocompromised individuals. It has been reported that greater than 70% of all AIDS patients in the U.S. develop pneumonia complications due to P. carinii (W. T. Hughes, J. Protozool. 38:243S (1991); J. Mills, Rev. Infect. Dis. 8:1001 (1986)) and that 50% of AIDS patients have recurrent infections (L. J. Deloreno, et al. Chest 88:79-83 (1985); H. Masur, and J. A. Kovacs, Infect. Dis. Clin. N. Am. 2:419-428 (1988)). Reports indicate that P. carinii infection is now recognized as a major cause of death (J. F. Murry, et al., Am. Rev. Respir. Dis. 135:504-509 (1987)) in AIDS patients in spite of the introduction of prophylaxis for treatment of the pneumonia (W. T. Hughes, J. Protozool. 38:2S (1991). Furthermore, there is a high incidence of adverse reactions among AIDS patients to existing anti-P. carinii treatments.
The worldwide incidence of P. carinii is difficult to determine because surveillance of the organism is not required in most countries (M. T. Cushion et al., Interntl. Rev. of Cyt. 131:59-106 (1991)). However, a review of worldwide reports of pneumonia showed that very few countries have not reported P. carinii (W. T. Hughes, in Pneumocystis carinii Pneumonitis W. T. Hughes, Ed. (CRC Press, Boca Raton, 1987), vol. 1, pp. 14-20, 97-104).
Analysis of P. carinii at the molecular level is in the very early stages. The genome is about 7000 kb in size and has at least 13 chromosomes (B. Lundren at al., Infect. Immun. 58:1705-1710 (1990); S. T. Hong, et al., J. Clin. Microbiol. 28:1785-1795 (1990)). The cloning and characterization of only a few genes has been reported. These data have revealed that the genome is A+T rich (U. Edman et al., Proc. Natl. Acad. Sci. U.S.A. 86:6503 (1989); J. C. Edman et al., Proc. Natl. Acad. Sci. U.S.A. 86:8625 (1989); L. D. Fletcher et al., Gene 129:167-174 (1993)). Little if any information is available on the regulatory sequences involved in transcription and translation. However, the greatest stumbling block to date in studying P. carinii has been the inability to reliably propagate sufficient quantities of the organism in the laboratory (E. Sloand, et al., J. Euk. Microbiol. 40:188-195 (1993)).
Rat derived and human derived P. carinii are very similar organisms, with differences at the DNA sequence level. Currently, a time consuming in vivo method is used whereby an immunocompromised mouse or rat is injected intratracheally with either rat-derived or human-derived P. carinii and the pathogen multiplies and develops in the lung tissues (E. Sloand, et. al., J. Euk. Microbiol. 40:188-195 (1993), K. K. Sethi, Experientia 48:63-67 (1992)).
These difficulties present a need for alternative strategies for study of the organism which can yield new therapeutic agents to prevent or control infection. In this regard, there is a need to identify molecular targets of pneumocystis whose function can be specifically inhibited by antimicrobial agents.
The design of effective antibiotics should exploit the biological differences between the pathogen and host. Designing new antibiotics requires the identification of potential targets in Pneumocystis carinii. The search for exploitable differences in the enzymatic pathways of P. carinii and humans is hindered by the limited understanding of the organism's biology. The use of in vitro methods to produce and study target enzymes of P. carinii and the use of tester strains allows an approach to developing new drugs against P. carinii without having to culture the living organism.
Because the amino acid sequences of the tRNA synthetases have diverged over evolutionary time, significant differences exist between the structures of the enzymes from mammals (e.g., human, bovine) and mammalian pathogens. These differences can be exploited by finding inhibitors of aaRS activity which specifically target a tRNA synthetase of a pathogenic organism, and which may further have specific antimicrobial activity. By selectively inactivating one or more of its aminoacyl-tRNA synthetases, Pneumocystis carinii infection can be controlled.